Methods of stimulating the immune system

ABSTRACT

This invention relates to a compound or group of compounds present in an active principle derived from plants of species  Euphorbia peplus, Euphorbia hirta  and  Euphorbia drummondii,  and to pharmaceutical compositions comprising these compounds. Extracts from these plants have been found to show selective cytotoxicity against several different cancer cell lines. The compounds are useful in effective treatment of cancers, particularly malignant melanomas and squamous cell carcinomas (SCCs). In a preferred embodiment of the invention, the compound is selected from the group consisting of jatrophanes, pepluanes, paralianes and ingenanes, and pharmaceutically-acceptable salts or esters thereof, and more particularly jatrophanes of Conformation II.

[0001] This invention relates to a compound or group of compoundspresent in an active principle derived from the family Euphorbiaceae,and in particular in plants of the species Euphorbia peplus, Euphorbiahirta and Euphorbia drummondii. Extracts from these plants have beenfound to show selective cytotoxicity against several different cancercell lines. Compounds present in the sap of Euphorbia spp. are useful ineffective treatment of cancers, particularly malignant melanomas andsquamous cell carcinomas (SCCs).

BACKGROUND OF THE INVENTION

[0002] There is a strong association between exposure of the skin to theultraviolet light component of sunlight and the development of skincancers, such as malignant melanoma and the non-melanoma skin cancers,mainly basal cell carcinomas (BCCs) and squamous cell carcinomas (SCCs).The incidence of these cancers has been rapidly increasing world wide.In Britain, there were 4000 newly-diagnosed cases of malignant melanomain 1994, an 80% increase over the past 10 years (Wessex Cancer Trust,1996). In the United States, approximately 34,100 new cases wereexpected, an increase of 4% per year. Queensland, Australia, has thehighest incidence of melanoma in the world, but early detection andwidespread public health campaigns and the promotion of the use ofsunscreens and reduction of ultraviolet exposure have helped to reducethe number of deaths. BCCs currently affect one in 1,000 in the U.K.population, and the incidence has more than doubled in the last 20 years(Imperial Cancer Research Fund, U.K., 1997). One million new cases ofBCCs and SCCs are expected to be diagnosed in the USA in 1997, comparedto 600,000 in 1990 and 400,000 in 1980 (National Oceanic and AtmosphericAdministration U.S.A., 1997). In Australia, there is no reason tosuspect that a similarly increasing incidence would not also apply,despite extensive publicising of the dangers of solar and UV radiation,with the Queensland population being at the greatest risk.

[0003] Over 90% of all skin cancers occur on areas of the skin that havebeen regularly exposed to sunlight or other ultraviolet radiation, withU.V.B. responsible for burning the skin and associated with malignantmelanomas, and U.V.A. associated with premature skin aging and thedevelopment of BCCs and SCCs (Wessex Cancer Trust, 1996). Childhood sunexposure has been linked to the development of malignant melanoma inyounger adults. Other risk factors include a genetic predisposition(fair complexion, many skin moles), chemical pollution, over-exposure toX-rays, and exposure to some drugs and pesticides. Depletion of theozone layer of the stratosphere is considered to contribute to long-termincreases in skin cancer.

[0004] Surgical removal is by far the most common treatment formalignant melanomas, BCCs and SCCs. This can take the form ofelectrodesiccation and curettage, cryosurgery, simple wide excision,micrographic surgery or laser therapy. Other treatments, used when thecancers are detected at a later stage of development, are externalradiation therapy, chemotherapy or to a lesser extent bio-immunotherapyor photodynamic therapy. The choice of treatment is dependent on thetype and stage of the disease and the age and health of the patient(National Cancer Institute, U.S.A., 1997).

[0005] All of the present treatments suffer from severe limitations. Themajor concern is the poor recognition of cancerous cells at the site ofexcision and the high likelihood of recurrence, necessitating follow-upsurgery and treatment, with the risk of further disfigurement andscarring. In one publication, the reported rates forincompletely-excised BCCs was 30-67% (Sussman and Liggins, 1996). Immunesuppression associated with surgery may cause any remaining cells toproliferate, and increase the risk of metastases. In melanoma patientsthere is a high risk that the cancer has already metastasized at thetime of initial surgery, and late recurrence leading to death is common.Present alternatives to surgery, such as radiation therapy andchemotherapy, also carry risks of immune suppression and poorspecificity. Immunotherapy and gene therapy hold the greatest promise,but the rational application of these is likely to be still decadesaway.

[0006] When the tumour is past the stage amenable to surgery, the mostcommon treatment for melanoma or metastatic skin cancer of all types ischemotherapy, which has been largely unsuccessful (Beljanski andCrochet, 1996)

[0007] In theory, an ideal drug would be one that when applied topicallyto an exposed melanoma, BCC or SCC, selectively necrotises the tumourcells or induces them to undergo apoptosis, without causing damage tothe surrounding healthy skin cells. In practice, this has yet to beachieved. The drugs currently available are neither selective norpenetrative.

[0008] The lay public is also enamoured of the concept of topicalchemotherapy. There have been many documented “home remedies” for skincancer, which have had disastrous consequences, eg the use of bootpolish (Adele Green, Queensland Institute of Medical Research, pers.Comm.) The major danger is the production of scar tissue, underneathwhich the tumour cells continue to grow. An extract derived from plantsof the genus Solanum (kangaroo apple or devil's apple) and purportedlycontaining solasodine glycosides has been available in Australia as anon-prescription preparation treatment of sunspots and solar keratoses,under the name “Curaderm”. However the preparation was shown in a smallclinical trial against BCCs to be ineffective, with 14/20 patientsshowing persisting tumour on histological examination of tissue from thetreated site. In some cases, histological examination of the site oftreatment revealed malignant tissue embedded in scar tissue. The authorswarned against self-diagnosis and treatment, particularly with irritantsubstances (Francis et al, 1989).

[0009] However, anecdotal reports suggest that plant sap extracts arestill being used by the general public for the treatment of sunspots orsolar keratoses, with some success being claimed.

[0010] The sap of plants of the family Euphorbiaceae, particularly thegenus Euphorbia, has been used in the folk medicine of many countries.The genus was named after an early Greek physician in deference to itspurported medicinal properties (Pearn, 1987). only recently have some ofthese claims been investigated scientifically. The genus is enormouslydiverse, ranging from small, low-growing herbaceous plants to shrubs andtrees. Nearly all reports of activity of these plants and their extractsare anecdotal or derived from traditional medicine, and the nature ofthe preparations used is frequently either unknown or very poorlydescribed. Activity has been claimed against a huge variety ofconditions, ranging from warts, “excrescences”, calluses, “cheloidtumours”, corns, whitlows or felons, “superfluous flesh” and the like,to a variety of cancers (see, for example, Hartwell: Lloydia 1969 32153).

[0011] As part of the screening program for anti-cancer activity carriedout on 114,000 extracts from 35,000 terrestrial plant species carriedout by the United States National Cancer Institute, a number of speciesof Euphorbia were tested. An aqueous suspension, an olive-oilsuspension, an alcohol extract and an acid extract were screened foractivity against the transplantable tumour cell line sarcoma 37. Fourspecies were tested. Of these, Euphorbia peplus showed no activity inany of the extracts; Euphorbia drummondii, Euphorbia pilulifera, andEuphorbia resinifera showed weak activity of an acid extract, an alcoholextract, and an olive-oil suspension respectively (Belkin andFitzgerald, 1953). A review of the scientific and medical literature ofthe past five years revealed a diversity of powerful active principlessuch as di- and tetra-terpenes, flavonoids, sterols and proteins in thisgenus, and many bioactive effects have been reported, with both positiveand adverse effects noted. These reports are summarized in Table 1. Inparticular the genus Euphorbia is well known to produce tumour promoterssuch as phorbol esters (Hecker, E.: “Cocarcinogens from Euphorbiaceaeand Thymeleaceae” in “Symposium on Pharmacognosy and Phytochemistry”(Wagner et al, eds., Springer Verlag 1970 147-165)). TABLE 1 SpeciesActive principle Action Reference Euphorbia whole plant: prostatic andlung Oksuz, S. aleppica aleppicatines, neoplasms et al (1996) diterpenepolyesters, cycloartene triterpenes, scopoletin, kaempferol, 4-hydroxybenzoic acid Euphoriba cerebrosides ? Falsone G biglandulosa etal (1994) Desf. Euphorbia latex skin irritant and Gundidza, bougheiitumour promoting M. et al effect (1993) Euphorbia latex: lipase homologyMoulin, A. characias (43.5%) with B et al (1994) chain of ricinEuphorbia whole plant: skin irritant Gundidza, cooperei phorbol ester M.et al NE Br (1992) Euphorbia alkaline extract treatment of Liu Y,fisheriana epilepsy et al (1994) Euphorbia whole plant inhibition ofVijaya, K, hirta bacteria of Shigella et al (1995) spp Euphorbia wholeplant: antidiarrhoeic Glavez, J. hirta flavonoid activity et al (1993)Euphorbia whole plant: ? Yoshida, T. humifusa hydrolysable et al (1994)tannins, polyphenol glucoside Euphorbia root: Chinese herbal Guo, Z.hylonoma 3,3′,4-tri-O-met- medicine ?? action et al (1995) methylellagicacid, betasitosterol Euphorbia whole plant: stimulation of Matsumoto,kansui ingenols expression of the T. et al macrophage Fc (1992) receptorEuphorbia pelletised plant rodenticide Gassling and lathyris materialLandis (1990) U.S. Pat. No. 4906472 Euphoriba latex mitogenic lectinStirpe, F. marginata et al (1993) Euphoriba ? quercetin Folk remediesfor Weedon and peplus hyperoside, warts, corns, Chick kaempferol,asthma, rodent (1976) and sitosterol, alkaloids, ulcer, BCC referencesglycosides cited therein Euphorbia diterpenes selectively cytotoxicFatope, poisonii for human kidney M. O. et al carcinoma cell (1996) lineA-498 Euphorbia latex inhibition of Jurberg, P. splendens mollusc (1995)Biomphalaria glabrata (vectors of schistosomiasis) Euphorbia whole plantreduces EBV- Imai, S. tirucalli specific cellular (1994) immunity inBurkitt's lymphoma

[0012] The most intensively studied species of this group is Euphorbiapilulifera L (synonyms E. hirta L. ; E. capitata Lam.), whose commonnames include pill-bearing spurge, snake-weed, cat's hair, Queenslandasthma weed and flowery-headed spurge. The plant is widely distributedin tropical countries, including India, and in Northern Australia,including Queensland. According to the “Encyclopedia of Common NaturalIngredients Used in Food, Drugs and Cosmetics” (Leung and Foster, 1996),the whole flowering or fruiting plant is used in herbal remedies,principally for cough preparations, and in traditional medicine fortreatment of respiratory conditions such as asthma, bronchitis, coughsand hayfever. This reference reports the active constituents ofEuphorbia pilulifera to be choline and shikimic acid, and that othercompounds present include triterpenes, sterols, flavonoids, n-alkanes,phenolic acids, L-inositol, sugars and resins. Of these components,shikimic acid is an essential intermediate in the synthesis of aromaticamino acids, and has been reported to have carcinogenic activity in mice(Evans and Osman, 1974; Stavric and Stoltz, 1976). Jatrophanes,ingenanes, and a tetracyclic diterpene designated pepluane wereidentified in the sap of Euphorbia peplus by Jakupovic et al (1998a) .The jatrophanes were stated to have a different conformation from thoseof previously-known jatrophanes. Jatrophanes are also stated to belongto a group of non-irritant diterpenes, which could have accounted totheir being overlooked in previous studies. There is no disclosure orsuggestion at all of any biological activity of the jatrophanes or ofthe new pepluane compound; nor is it suggested that any of thesecompounds might be useful for any pharmaceutical purpose.

[0013] A recent report describes selective cytotoxicity of a number oftigliane diterpene esters from the latex of Euphorbia poisonii, ahighly-toxic plant found in Northern Nigeria, which is used as a gardenpesticide and reputed to be used in homicides. One of these compoundshas a selective cytotoxicity for the human kidney carcinoma cell lineA-498 more than 10,000 times greater than that of adriamycin (Fatope etal, 1996).

[0014] In a series of patent applications, Tamas has claimed use ofEuphorbia hirta plants and extracts thereof for a variety of purposes,including tumour therapy (EP 330094), AIDS-related complex and AIDS(HU-208790) and increasing immunity and as an antifungoid agent fortreatment of open wounds (DE-4102054).

[0015] Thus, while there are isolated reports of anti-cancer activity ofvarious Euphorbia preparations (see Fatope et al, 1996; Oksuz et al,1996), not only are the compounds present in at least one Euphorbiaspecies reported to be carcinogenic (Evans and Osman, 1974; Stavric andStolz, 1976; Hecker, 1970; 1977), but at least one species has askin-irritant and tumour-promoting effect (Gundidza et al, 1993), andanother species reduces EBV-specific cellular immunity in Burkitt'slymphoma (Imai, 1994).

[0016] To our knowledge, there has been no reliable or reproduciblereport of the use of any extract from Euphorbia species for thetreatment of malignant melanoma or SCCs. An anecdotal report of hometreatment of a BCC with the latex of Euphorbia peplus (petty spurge ormilk weed) was the publication of Weedon, D. and Chick, J., entitled“Home treatment of basal cell carcinoma” (1976). The authors stated thatmedicinal propeties have been claimed for the milky juice of this plantsince the time of Galen, and it was widely used as a home remedy forcorns, warts, and asthma. At the turn of the century it was used by somephysicians in Sydney for the treatment of rodent ulcers. The author'spatient claimed to have treated himself over many years for multipleBCCs.

[0017] “The patient, a 54 year old male, had been seen sporadically atthe Royal Brisbane Hospital since 1971. On one visit he was noted tohave a clinical basal cell carcinoma on the anterior part of his chestwhich was confirmed by biopsy of a tiny specimen taken from one edge.Some days later when the biopsy site had healed the patient applied thesap of Euphorbia peplus every day for 5 days. The area becameerythematous and then pustular, after which the lesion sloughed off. Onhis return 6 weeks after treatment, the patient agreed to let ussurgically excise the small area of residual scarring. Multiple sectionsshowed dermal scar tissue which contained a few chronic inflammatorycells, but showed no evidence of residual tumour.”

[0018] The authors stated that “this communication should in no way betaken as a recommendation of the form of therapy”. There are a fewreports cautioning on the corrosive nature of the sap, and minor eyedamage that has resulted from the home treatment of warts usingEuphorbia peplus (Eke, T., 1994). It appears likely that the effectreported by Weedon and Chick resulted from the irritant activity of theEuphorbia peplus sap, and that, as in the case of the Solanum extract“Curaderm” reported by Francis et al (1989), there is a high risk ofresidual tumour cells surviving in or under the scar tissue that resultsfrom such treatment.

[0019] The inventor has now surprisingly found that sap of plants fromthree different Euphorbia species, Euphorbia peplus, Euphorbia hirta andEuphorbia drummondii, specifically inhibits growth of three differenthuman tumour cell lines, including malignant melanoma. Moreover, at verylow concentrations, sap from Euphorbia peplus and Euphorbia hirtainduced differentiation of malignant melanoma cells so that they adoptedthe morphological appearance of normal melanocytes. At similar or evenlower concentrations an extract stimulated activation of themetallothionein gene promoter and expression of a reporter gene in MM96Lmalignant melanoma cells. The results were particularly striking, sincethe melanoma cell line which was used is refractory to inhibition by allof the conventional chemotherapeutic agents which have been testedagainst it (Maynard and Parsons, 1986).

SUMMARY OF THE INVENTION

[0020] In a first aspect, the invention provides a compound or compoundspresent in plants of the genus Euphorbia, and in particular in sap ofEuphorbia peplus, Euphorbia hirta and/or Euphorbia drummondii, which:

[0021] (a) is able to kill or inhibit the growth of cancer cells, butdoes not significantly affect normal neonatal fibroblasts, orspontaneously transformed keratinocytes;

[0022] (b) has activity which is not destroyed by heating at 95% for 15minutes;

[0023] (c) has activity which is not destroyed by treatment withacetone;

[0024] (d) has activity which can be extracted with 95% ethanol; and

[0025] (e) stimulates metallothionein gene activation.

[0026] Preferably, the compound(s) is able to inhibit the growth of atleast one cell line selected from the group consisting of MM96L, MM229,MM220, MM237, MM2058, B16, LIM1215, HeLa, A549, MCF7, MCC16 and Colo16as herein defined. More preferably, the compound(s) is able to inhibitgrowth of or to induce differentiation in MM96L cells.

[0027] Even more preferably the compound is also able to induce normalmelanocytes to proliferate.

[0028] Preferably, the compound is present in sap of E. peplus or E.hirta .

[0029] It will be clearly understood that while the invention isdescribed in detail with reference to compounds detected in sap or sapextracts, these compounds, when present in or extracted from wholeplants or parts thereof, are still within the scope of the invention.

[0030] In a second aspect, the invention provides a compositioncomprising an active compound as described above, together with apharmaceutically-suitable carrier or diluent.

[0031] More preferably the compound is selected from the groupconsisting of jatrophanes, pepluanes, paralianes and ingenanes.

[0032] Where the compound is a jatrophane, it is preferably ofConformation II as defined by Jakupovic et al (1998a). It will beclearly understood that the substitutions observed innaturally-occurring jatrophane, pepluane and paraliane skeletons arewithin the scope of the invention. These include the followingsubstitutions and analogues.

[0033] Compounds of this type have been found in a variety of plants ofthe genus Euphobia (Jakupovic et al, 1998a, b, c; Marco et al, 1998).TABLE 2 Natural Substitutions Observed for the Jatrophane, Pepluane andParaliane Skeletons. (Jakupovic et al, 1998a, b, c; Marco et al, 1998)Carbon position Jatrophane Pepluane Paraliane 1 H, OAc H₂, OAc H & OAc,H₂, 2 OAc & H, CH₃ & OAc, CH₃ CH₃ & H CH₃ & H, CH₃ & H & OAc 3 OH, OAc,OiBu, OCinn, OBz, OBz OBz OBzOCH₂CO, PhCO₂CH₂CO₂ 4 H H H 5 OAc, OiBu,Omebu, OAcOAc OAc OAc 6 exocyclic double bond CH₃ , CH₃ , CH₂OAc CH₂OAc7 H₂, OAc, OiBu, OMeBu, OPr, H₂, H₂, OCOiPr, OCOEt 8 H₂, OH, OAc, OiBu,OMebu, OAc, double H, OAc OBz, OAng, bond to C12 9 OH, OAc, OCinn, ONic,═O OAc, 9-18 ═O double bond 10  (CH₃)₂ CH₃ & OAc, (CH₃)₂ double bond to11, CH₃ 11  double bond to 12 H₂, double H₂ bond to 10 & OH 12  doublebond to 11 H, double H bond to 8 13  CH₃ CH₃ CH₃ 14  H & OH, H & OAc, ═OOAc OAc 15  OAc, OH OH OH 18  H, H₂,

[0034] Even more preferably, the compound is selected from the groupconsisting of:

[0035] 5,8,9,10,14-pentaacetoxy-3-benzoyloxy-15-hydroxypepluane(pepluane);

[0036] 15-pentaacetoxy-9-nicotinoyloxy-14-oxojatropha-6(1),11E-diene(jatrophane 1);

[0037]2,5,7,9,14-hexaacetoxy-3-benzoyloxy-15-hydroxy-jatropha-6(17),11E-diene(jatrophane 2);

[0038]2,5,14-triacetoxy-3-benzoyloxy-8,15-dihydroxy-7-isobutyroyloxy-9-nicotinoyloxyjatropha-6(17),11E-diene (jatrophane 3);

[0039]2,5,9,14-tetraacetoxy-3-benzoyloxy-8,15-dihydroxy-7-isobutyroyloxyjatropha-6(17),11E-diene)(jatrophane 4);

[0040]2,5,7,14-tetraacetoxy-3-benzoyloxy-8,15-dihydroxy-9-nicotinoyloxyjatropha-6(17),11E-diene(jatrophane 5);

[0041]2,5,7,9,14-pentaacetoxy-3-benzoyloxy-8,15-dihydroxyjatropha-6(17),11E-diene(jatrophane 6);

[0042] 20-acetyl-ingenol-3-angelate;

[0043] and pharmaceutically-acceptable salts or esters thereof.

[0044] In one preferred embodiment of the invention, the compositionadditionally comprises β-alanine betaine hydrochloride ort-4-hydroxy-N,N-dimethyl proline.

[0045] In a third aspect, the invention provides a method of treatmentof a cancer, comprising the step of administering an anti-cancereffective amount of a compound of the invention to a mammal in need ofsuch treatment.

[0046] Preferably, the cancer is a solid tumour. More preferably, thecancer is selected from the group consisting of malignant melanoma,other skin cancers including Merkel cell carcinoma, squamous cellcarcinoma and basal cell carcinoma, lung cancer, colon cancer, prostatecancer, cervical cancer and breast cancer.

[0047] In a fourth aspect, the invention provides a method of inhibitingproliferative activity of neoplastic cells, comprising the step ofexposing the cells to an anti-proliferative amount of a compound of theinvention. The cells may be treated either ex vivo or in vivo.

[0048] In a fifth aspect, the invention provides a method of preventingor alleviating damage to skin, caused by ultraviolet irradiation,ionizing radiation, microwave radiation, exposure to ozone, or the like,comprising the step of topically administering an effective amount of acompound of the invention to a subject in need of such treatment. Thisaspect of the invention may be used in the treatment of solar keratosis,skin damage occurring during radiotherapy, and the like.

[0049] In a sixth aspect the invention provides a method of stimulatingproliferation of non-neoplastic cells comprising the step of exposingthe cells to a proliferation-inducing amount of a compound or acomposition of the invention. This is useful in inducing regeneration oftissues and, because T-lymphocytes proliferate in response to thecompositions of the invention, is useful in promoting the immuneresponse to disease states.

[0050] The mammal may be a human, or may be a domestic or companionanimal. While it is particularly contemplated that the compounds of theinvention are suitable for use in medical treatment of humans, it isalso applicable to veterinary treatment, including treatment ofcompanion animals such as dogs and cats, and domestic animals such ashorses, cattle and sheep, or zoo animals such as felids, canids, bovids,and ungulates.

[0051] The compounds and compositions of the invention may beadministered by any suitable route, and the person skilled in the artwill readily be able to determine the most suitable route and dose forthe condition to be treated. Dosage will be at the discretion of theattendant physician or veterinarian, and will depend on the nature andstate of the condition to be treated, the age and general state ofhealth of the subject to be treated, the route of administration, andany previous treatment which may have been administered.

[0052] The carrier or diluent, and other excipients, will depend on theroute of administration, and again the person skilled in the art willreadily be able to determine the most suitable formulation for eachparticular case. It is contemplated that compounds of the invention maybe administered orally, topically, and/or by parenteral injection,including intravenous injection.

[0053] Methods and pharmaceutical carriers for preparation ofpharmaceutical compositions are well known in the art, as set out intextbooks such as Remington's Pharmaceutical Sciences, 17th Edition,Mack Publishing Company, Easton, Pa., USA.

[0054] For the purposes of this specification it will be clearlyunderstood that the word “comprising” means “including but not limitedto”, and that the word “comprises” has a corresponding meaning.

BRIEF DESCRIPTION OF THE FIGURES

[0055]FIG. 1 shows the effect of E. peplus sap on metallothionein geneactivation, measured by detecting the activity of β-galactosidase usinga chromogenic substrate.

[0056]FIG. 2 shows the proliferation of MCF7 breast cancer cells grownin microtitre wells in the presence of E. peplus sap for 7 days(expressed as a percentage of control values).

[0057]FIG. 3 shows the absorbance profile at 195 nm, following RP-HPLCsub-fractionation of an ethanol-soluble extract of E. peplus sap.

[0058]FIG. 4 shows the results of repeated RP-HPLC chromatography offraction 14 from FIG. 3.

[0059]FIG. 5 shows the constant diode array spectrum of the peak fromFIG. 4.

[0060]FIG. 6 shows the results of treatment of MM96L melanoma cells withFraction 15 from Example 7. Cells are stained with antibody TRP-1,directed against the cytoskeleton A,,B: 4 days, C,D: 21 days

[0061]FIG. 7 shows the results of thin layer chromatography of theether-soluble fraction from Example 6 using chloroform:ethyl acetate(82:18) as the developing solvent.

[0062]FIG. 8 shows the results of further purification by 2-dimensionalTLC on silica gel, using hexane:ethyl acetate (1:1) in the firstdimension, and toluene:acetone (9:1) in the second dimension.

[0063] A: Spots 34-45 were visualised on a UV light box. Activities werescored towards MM96L at a 1 in 500 dilution (+++=no effect, −=completecell death, d=100% reversion of cells to a dendritic appearance.

[0064] B: Spots 14-20 were visualised on a UV light box. Activities werescored towards MM96L at a 1 in 500 dilution (+++=no effect, −=completecell death, d=100% reversion of cells to a dendritic appearance.

[0065] C: Spots 21-27 were visualised on a UV light box. Activities werescored towards MM96L at a 1 in 500 dilution (+++=no effect, −=completecell death, d=100% reversion of cells to a dendritic appearance.

[0066]FIG. 9 shows results of ascending chromatography of crude sap onHPTLC using a toluene:acetone (9:1) solvent system. Opaque bands 1-7were visualised on a UV light box

[0067]FIG. 10 shows results of ascending chromatography of fraction 1from FIG. 9 on HPTLC using a hexane:ethyl acetate (4:1) solvent system.Bands A-G were visualised on a UV light box. (Side strip stained with0.1% iodine in chloroform revealed Fraction G—inactive against MM96L).

[0068]FIG. 11 shows results of ascending chromatography of fraction 1from FIG. 9 on HPTLC using a hexane:ethyl acetate (4:1) solvent system.Band H was visualised on a UV light box.

[0069]FIG. 12 shows the results of ascending chromatography of diethylether soluble fraction prepared from crude sap on preparative thin layerchromatography (PLC, Merck) using hexane:ethyl acetate (4:1) solventsystem. Zones H and A-F were visualised on a UV light box, extracted,and used for in vivo studies.

[0070]FIG. 13 shows the results of treatment of subcutaneous humanmelanoma MM96L xenografts in nude mice with a partially purifiedfraction prepared as described in Example 11. Arrows denote the positionof topical treatments for a tumour (right-hand side) and for normal skin(top of back) There was no evidence of residual tumour growth or lastingdamage to the normal skin 32 days after the treatment regimen began, and20 days after the first topical application.

DETAILED DESCRIPTION OF THE INVENTION

[0071] The invention will be described in detail by reference only tothe following non-limiting examples and to the figures.

Example 1 Inhibitory Activity of Euphorbia Sap Against Tumour Cell Lines

[0072] The ability of sap of three Euphorbia species, Euphorbia peplus,Euphorbia hirta and Euphorbia drummondii to inhabit the growth of threedifferent human tumour cell lines was tested. The activity againstnormal skin fibroblasts was tested as a control.

[0073] The cell lines were maintained in RPMI medium containing 5%foetal calf serum (FCS), and assays were performed in the same medium.

[0074] Sap was collected from plants growing randomly on cultivated soilon a farm at Palmwoods, in the Sunshine Coast hinterland, South-EastQueensland. The plant stem surface was briefly washed with 70% ethanol,and scissors washed in ethanol were used to cut the stem and release themilky latex sap. The sap was collected into 10 ml sterile plasticcentrifuge tubes, transported at 4° C. to Brisbane and stored frozen at−20° C. Prior to use, the sap was serially diluted Five-fold up to 1 in3125 into sterile 1.5 ml Eppendorf tubes using sterile MilliQ water. 10μL aliquots of each dilution were added to each two of microtitre platewells containing 100 μl of the cell lines. Assays were performed induplicate.

[0075] After 5 days, cells were examined blind, for inhibition of growthcompared to control untreated cell samples. The results are summarizedin Tables 3 to 6, in which the cell lines tested were

[0076] NFF normal skin fibroblasts

[0077] MM96L malignant melanoma, brain metastasis

[0078] HeLa cervical cancer

[0079] HACat spontaneously-transformed human keratinocytes

[0080] and the scale is 0=no effect to 5=complete cell death

[0081] The dilution in the table heading refers to the dilution of thesample before addition to the culture. Thus, the dilution in the finalculture is approximately 10-fold greater. TABLE 3 NFF Normal FibroblastsDilution Sample 1 Sample 2 Sample 1/5 1/25 1/125 1/625 1/3125 1/5 1/251/125 1/625 1/3125 E. peplus 3 2 0 0 0 0 0 0 0 0 E. hirta 5 0 0 0 0 0 00 0 0 E. drummondii 4 0 0 0 0 0 0 0 0 0 No addition 0 0 0 0 0 0 0 0 0 0

[0082] TABLE 4 MM96L Malignant Melanoma Dilution Sample 1 Sample 2Sample 1/5 1/25 1/125 1/625 1/3125 1/5 1/25 1/125 1/625 1/3125 E. peplus5 4 4 0 0 5 3 1 0 0 E. hirta 5 4 1 0 0 4 1 0 0 0 E. drummondii 5 2 1 0 05 2 0 0 0 No addition 0 0 0 0 0 0 0 0 0 0

[0083] TABLE 5 Hela Cells Dilution Sample 1/5 1/25 1/125 1/625 1/3125 E.peplus 5 3.5 3 1 1 E. hirta 5 5 5 5 0 E. drummondii 5 0 0 0 0 Noaddition 0 0 0 0 0

[0084] TABLE 6 HACat keratinocytes Dilution Sample 1/5 1/25 1/125 1/6251/3125 E. peplus 4 0 0 0 0 E. hirta 5 0 0 0 0 E. drummondii 5 0 0 0 0 Noaddition 0 0 0 0 0

[0085] From these results it can be seen that:

[0086] a) E. peplus was active against HeLa cells, and to a lesserextent against MM96L cells.

[0087] b) E. hirta was active against MM96L cells and very stronglyactive against HeLa cells.

[0088] c) E. drummondii had a lesser effect against MM96L than the othertwo samples, and inhibited HeLa cells only at the highest concentrationtested.

[0089] d) NFF normal fibroblasts were severely affected at the 1/5dilution, but only mildly affected at the other dilutions. For example,at a dilution of 1/25, there was mild inhibition of NFF cells (rating2), but severe inhibition of MM96L cells (rating 4). At a dilution of1/125, no effect was observed against NFF cells (rating 0) , but severeinhibition of MM96L cells (rating 4) was observed for one sample, andmilder inhibition (rating 1) with the duplicate sample). HACat cells,which could be considered as representative of normal keratinocytes,were only inhibited at the highest concentration.

[0090] At high concentrations of E. peplus sap, it appeared that therewas direct killing of MM96 cells. However, at lower concentrations (downto a dilution of 1/625), although no growth inhibition was observed, thesurviving cells were dendritic, and had the appearance of normalmelanocytes. Without wishing to be limited to any proposed mechanism, itappears that E. peplus sap may contain at least one agent which promotesdifferentiation, rather than directly cytotoxic agents which damage DNA.

Example 2 Effect of Heat or Acetone Treatment on Activity of EuphorbiaSap

[0091] The experiment described in Example 1 was repeated for E. peplusand E. hirta by a different person, using different cell linepreparations, different plant samples and a different rating scale.

[0092] The samples were either prepared as described in Example 1, orwere subjected to treatment with heat or acetone. Undiluted extracts ofplant sap were heated at 95° C. for 15 minutes. For the acetonetreatment, 40 μl extract was suspended in 400 μl acetone, and the tubeshaken on a vortex mixer. Contents were centrifuged at 10,000 g for 3minutes and the supernatant (acetone-soluble fraction) removed to aseparate tube. Both the pellet and supernatant were left in open tubesat room temperature in the fume hood overnight with exhaust fanoperating to evaporate the residual acetone.

[0093] The results are shown in Tables 7 to 9, in which +++ indicates noeffect, and − indicates 100% cell death. “C” indicates that the culturewas contaminated. Using this rating scale the results were even morestriking than in Example 1, with strong inhibitory activity beingobserved up to a dilution of 1:3125. However, some growth inhibition ofNFF cells was seen in this experiment.

[0094] Neither heat nor acetone affected the anti-tumour activitysignificantly. With acetone treatment, most activity was found in thepellet, particularly in the case of E. hirta, though some activity wasalso present in the soluble fraction. This suggests that the compoundsresponsible are not protein in nature, and that at least one componentmay be a lipid. TABLE 7 MM96L Dilution Sample 1 Sample 2 Sample 1/5 1/251/125 1/625 1/3125 1/5 1/25 1/125 1/625 1/3125 E. peplus − − ± ± ± − −± + + E. hirta − ++ ++ +++ +++ E. hirta heat ± + + +++ +++ acetonesoluble ± + + +++ +++ E. peplus acetone soluble ± ++ +++ +++ +++ E.hirta acetone − + + +++ +++ precipitate E. peplus acetone − −/± ++ ++++++ − − ++ +++ +++ precipitate E. hirta E. peplus heat − + +/++ +/+++/++ − + ++ ++ ++

[0095] TABLE 8 NFF Dilution Sample 1 Sample 2 Sample 1/5 1/25 1/1251/625 1/3125 1/5 1/25 1/125 1/625 1/3125 E. peplus − + c +/++ ++ − c +++ ++ E. hirta − + + + ++ − + ++ ++ ++ E. hirta heat + + ++ ++ ++acetone soluble ± ++ ++ ++ ++ E. peplus acetone soluble + ++ ++ ++ ++ E.hirta acetone ± + + ++ ++ precipitate E. peplus acetone − ± + ++ +precipitate E. hirta E. peplus heat − + ++ + ++

[0096] TABLE 9 HeLa cells Sample 1/5 1/25 1/125 1/625 1/3125 E. peplus± + +++ +++ +++ E. hirta − +++ +++ +++ +++ E. hirta heat + ++ +++ ++++++ acetone +++ +++ +++ +++ +++ soluble E. peplus acetone +++ +++ ++++++ +++ soluble E. hirta acetone ppte ++ ++ +++ +++ +++ E. peplusacetone ppte ± +++ +++ +++ +++ E. hirta E. peplus heat − +++ +++ +++ +++

Example 3 Further Tests Using E. peplus

[0097] Since E. peplus is the most abundant of the three plants testedin these studies, further experiments utilised extracts from thisspecies. This is not to be taken to imply that activity is not presentin the other two species.

[0098] Example 2 was repeated, using MM229 and MM220 human malignantmelanoma cells and B16 mouse malignant melanoma cell lines, in additionto NFF and MM96L cells. Assays were performed in duplicate, usingaddition of an equivalent amount of water as a control, and dilutions ofthe pellet and supernatant fractions after acetone treatment from 1/20to 1/12500. The results are summarised in Table 10. TABLE 10 DILUTIONH₂O Sample Control 1/20 1/100 1/500 1/2,500 1/12,500 1/20 1/100 1/5001/2,500 1/12,500 NFF pellet +++ + +++ +++ +++ +++ +/++ +++ +++ +++ +++NFF supernatant +++ + +++ +++ +++ +++ + +++ +++ +++ +++ MM96L pellet +++− + +/++ +++ +++ − ++ +++ +++ +++ MM96L +++ ± ++ ++ ++ ++ − + ++ +++ +++supernatant Hela pellet +++ − + ++/+++ +++ +++ − ++ ++ +++ +++ Hela +++− ± ++ ++ ++ − ± ++ +++ +++ supernatant MM229 pellet ++ − + + ++ ++ − +± ++ ++ MM229 ++ − + +/++ + ++ ++ + ++ + + supernatant MM220 pellet ++ −++ ++ ++ − + ++ ++ ++ ++ MM220 ++ − + ++ ++ −− − ++ ++ ++ ++ supernatantB16 pellet ++ − − ++ ++ − − ++ ++ ++ ++ B16 supernatant ++ ++ ++ ++ ++++ ++ ++ ++ ++ +30

[0099] The results confirm those obtained in Example 2. At a dilution of1/100 to 1/50 there was no effect on NFF cells, but significantinhibition of M96L cells was observed. The melanoma cells surviving atthese dilutions had the appearance of normal melanocytes. Inhibition ofthe other two human melanoma cell lines and of the mouse melanoma cellline was also observed.

[0100] Similar results were obtained using Merkel cell carcinoma (MCC16) or squamous cell carcinoma (Colo 6) cells. The results are shown inTable 11.

[0101] Dendritic cell morphology was displayed by squamous cellcarcinoma, even at 1 in 500,000 dilution. This extreme potency of thecrude extract was also evident for Merkel cell inhibition, which wasalso still evident at 1 in 500,000dilution. TABLE 11 Effect of crude sapfrom E. peplus on Merkel cell carcinoma (MCC16) and squamous cellcarcinoma (Colo16) cell numbers. Cell line Sample 1/50 1/500 1/5,0001/50,000 1/500,000 Colo16 Solvent +++ +++ +++ +++ +++ (control) crude E.peplus − ++d* +++d* +++d* +++d* sap MCC16 Solvent +++ +++ +++ +++ +++(control) crude E. peplus − ±* + + ++ sap

Example 4 Ethanol Extract of E. peplus

[0102] A fresh preparation of sap from E. peplus was subjected toextraction with 95% aqueous ethanol. Ethanol was removed from thesoluble fraction after extraction by vacuum centrifugation, and thefraction was reconstituted to its original volume in tissue culturemedium (RPMI1640) containing 5% foetal calf serum and antibiotics. Thepellet remaining after ethanol extraction was dried by vacuumcentrifugation and reconstituted to its original volume in tissueculture medium as described above. The crude sap (C), the solublefraction (S) and the pellet (P) were tested as described above againstNFF cells, the melanoma cell lines MM96L, MM-537, MM229 and MM2058, andalso against the colon cancer cell line LIM1215 and the lung cancer cellline A549. Assays were performed in triplicate, and were assessed afterfour days culture following addition of the sample. The results areshown in Table 12, in which + indicates normal appearance of cells, ++indicates a possible increase in cell numbers, and − indicates celldeath. TABLE 12 Dilution 1/20 1/100 1/500 1/2,5O0 Cell line C S P C S PC S P C S P NFF − +/− + + + + + + + + + + MM 96L − +/− +/− +/− +/− +/−+/− +/− +/− +/− + + MM 537 − − + +/− + + + + + + + + MM 229 − +/− ++/− + + + + + + + + MM 2058 − +/− +/− +/− + + + + + + + + Hela − +/−+/− + + + + + + + + LIM 1215 − − + − + + + + + + + ++ A 549 − − +/− +/−− +/− +/− +/− + +/− +/− +

[0103] The results obtained were consistent with those of the previousexperiments. Again at low doses the MM96L cells had a dendriticappearance. All of the tumour cell lines as well as the normalfibroblast cell line NFF were killed by the crude sap and by the solublefraction obtained by ethanol extraction at a dilution of 1/20. Itappeared that the majority of the activity partitioned to theethanol-soluble fraction. The lung cancer cell line A459 appeared to beparticularly susceptible, being affected at a dilution of up to 1/2500by both the crude sap and by the soluble fraction.

Example 5 Reporter Assay for Gene Expression in Transfected MM96LMalignant Melanoma Cell Line

[0104]E. peplus sap in phosphate-buffered saline diluent was added towells containing MM96L cells or the breast cancer cell line MCF7transfected with a construct consisting of the sheep metallothioneinpromoter, upstream of a β-galactosidase reporter gene which had beensubstituted for the metallothionein gene. The assay thus becomes ameasure of gene expression and in particular, of potentialtranscription, translation and expression of the metallothionein gene.Cells were treated with 4 extract in microtitre plates for 20 hr, 100 μMZnSO₄ was added and the plates incubated for a further 5 hr, and themedium was removed. β-galactosidase activity was then measured byincubation of the cells for 1-2 h at 37° C. with a chromogenicsubstrate. This assay is used as a sensitive test for transcriptionalactivation of genes.

[0105] The results are shown in FIG. 1.

[0106] This shows that there was a marked stimulation of metallothioneingene activation, as measured by increased β-galactosidase reporter geneexpression, which surprisingly became more evident as the sample furtherwas diluted. The mechanism by which E. peplus sap mediates this effectis not understood. Whereas known drugs specific for inhibition ofhistone deacetylase activity demonstrate increasing expression of thereporter gene with increasing concentration of drug, E. peplus exhibitsan inverse dose response. However, the results indicate that this assaycan be used to monitor purification of the active agents(s) in E. peplussap or the plant itself.

[0107] The metallothionein protein has antioxidant activity, and isimplicated in a protective role against heavy metal-induced cancers.Activation of the metallothionein promoter occurred at concentrations ofE. peplus sap too low to effect direct cell killing, except for theextremely sensitive breast cancer cell line MCF7 (FIG. 2). The change inappearance of MM96L melanoma cells to the dendritic morphology of normalmelanocytes at these dilutions possibly implicates the metallothioneingene in these effects.

Example 6 Subfractionation of Ethanol-Soluble Extract

[0108] The soluble fraction obtained by extraction with 95% ethanol,performed as in Example 4, was subjected to isocratic reverse-phasehigh-performance liquid chromatography (RP-HPLC).

[0109] 100 μl of crude extract was dissolved in 1 ml 95% ethanol andperiodically shaken at 4° C. overnight. The extract was centrifuged at10,000 x g for 4 minutes, and the supernatant was removed and dried byvacuum centrifugation. The solids were reconstituted in 100 μl runningbuffer centrifuged briefly, and the soluble material applied to aBrownlee Aquapore RP-300 column (C8), 220×4 mm, with a 30×4 mm RP-300guard column.

[0110] The running buffer was acetonitrile:water 50:50 (V/V), and theflow rate was 0.75 ml/mn. Fractions were collected at 0.5 min intervals,and the absorbance profile at 195 mn was monitored. The absorbanceprofile is shown in FIG. 3.

[0111] Fractions were dried by vacuum centrifugation, reconstituted in500 μl PBS, and assayed against MM96L. cells and in the metallothioneinreporter assay as described above. Fractions 13 to 28 all inducedcomplete reversion of MM96L cells to a dendritic appearance, but celldeath was not observed. The effect was much more striking in thereporter assay, in which activity was still observed at a dilution of1/10,000 (ie. at a final concentration in the culture of 1/100,000).

[0112] In addition to the foregoing results, the inventor has found thatfollowing ultracentrifugation, activity against MM96L cells is foundboth in the supernatant and in the pellet, and that activity cannot beremoved by passing a sample through a molecular weight cut-off membrane.In addition to the cell lines tested above, proliferation of cells ofthe MCF7 breast cancer cell line was inhibited by E. peplus sap at afinal dilution of up to 1/100,000. Cell numbers were assessed using thebicinchoninic acid reagent (Pierce). Results are shown in FIG. 2.

Example 7 Solvent Fractionation

[0113] Further solvent fractionation of the crude latex of E. peplus waseffected by a series of solvents of increasing polarity. To 1 ml crudelatex was added 20 ml diethyl ether in a centrifuge tube. The tube wasshaken and centrifuged at 5000 g for 5 minutes to partition the layers.The diethyl ether upper layer was removed and the procedure repeatedtwice. The ether fractions were combined, concentrated to dryness on arotary evaporator and reconstituted in 1 ml DME for bioassay. In asimilar manner, the residue was extracted with ethyl acetate, followedby methylene chloride. The initial ether extract obtained the majorityof the activity as measured by decrease in cell numbers of MCF7 breastcancer cells and reversion to a dendritic appearance. However, activitywas also demonstrated from the fractions derived from the ethyl acetateand methylene chloride layers. No activity was seen in the finalwater—soluble (aqueous) fraction. The results are summarised in Table13. TABLE 13 Cell line* Sample 1/50 1/500 1/5,000 1/50,00 1/500,000 NFFcrude E. peplus − ± + + + latex diethylether − ± + + + fraction ethylacetate ± + + + + fraction methylene + ± + + + chloride fraction aqueousfraction + + + + + HT144 crude E. peplus − − + + + latex diethylether −± + + + fraction ethyl acetate ± + + ++ ++ fraction methylene + + ++ ++++ chloride fraction aqueous fraction + ++ ++ ++ ++ MCF7 crude E. peplus− − ± ± ± latex diethylether − − ± ± ± fraction ethyl acetate − ± ± ± ±fraction methylene ± ± ± + + chloride fraction aqueous fraction + + + ++

[0114] CMV promoter activity was assayed in HeLa cells infected with arep-cation-deficient adenovirus construct, in which the Ela gene hadbeen replaced by the CMV promoter driving β-galactosidase. The results,shown in Table 14, are expressed as a percentage of the control valuesof infected, untreated cells. TABLE 14 Dilution Sample 1/50 1/5001/5,000 crude E. peplus latex 170 175 400 diethylether fraction 240 250345 ethyl acetate fraction 630 550 360 methylene chloride 746 420 170fraction aqueous fraction 180 100 100 solvent control* 100 approx 100100

[0115] The results obtained are qualitatively similar to those seen withother differentiation-inducing agents, such as histone deacetylaseinhibitors or butyrate, albeit with more potent activity than seen withthese agents. The lower promoter activity observed with the crude andthe diethylether extracts at higher concentrations probably reflectscell killing effects against HeLa cells seen at those concentrations.

[0116] In a further solvent fractionation experiment, the crude sap waspartitioned between methanol:water (17:3) and n-hexane, a solventpartition expected on the basis of previous reports to separatediterpenes (polar phase) from the triterpenes (heptane phase) (Evans andKinghorn 1977). Unexpectedly, however, activity was detected in bothphases, suggesting that the active principles behave anomalously in thissystem.

[0117] Another solvent fractionation approach was suggested by the needto clarify samples prior to HPLC analysis. The crude latex was mixedwith ethanol to 70-95% and shaken overnight at 4° C. The mixture wascentrifuged at 1,000 g for 10 min, and the supernatant was removed andconcentrated to approx one third the original volume of crude sap. Tothe concentrate was added 100% acetonitrile to 30-60%. The resultingwhite precipitate was removed by centrifugation at 12,000 g for 10minutes. The supernatant was enriched in macrocyclic diterpenes(jatrophanes and pepluane), as determined by TLC and mass spectroscopy.This observation points the way to a suitable large scale process forenrichment of the active principles

Example 8 Further Activity-Guided Subfractionation of theEthanol-Soluble Extract

[0118] Fractions 14 and 15 from the HPLC subfractionation described inExample 7 and FIG. 3 were further purified by repeated chromatography,selecting the dominant symmetrical peak with constant diode arrayspectra (eg. fractions 14 and 15; results for fraction 14 are shown inFIGS. 4 and 5). Activity of the purified fractions in causing reversionof MM96L to the dendritic appearance was confirmed by cell assay.

[0119] The features of the change to MM96L cells after the addition ofFraction 15 are shown in FIG. 6. Cells were visualised asphotomicrographs, using an antibody coupling procedure. The firstantibody, a mouse monoclonal directed towards tyrosinase-related protein1 (TRP-1) , was detected with a second antibody, sheepanti-mouse—alkaline phosphatase conjugate, using bromo-chloro-indolylphosphate and nitroblue tetrazolium (BCIP/NBT) as developing substrates.After four days of incubation (FIGS. 6A and 6B) there was a markedreduction in the number of melanoma cells and a pronounced change intheir morphology. The cells had reverted to a long, spindly (dendritic)appearance, characteristic of normal mature melanocytes. All cells inthe field appeared to have adopted this altered morphology, which issurprising given the heterogeneous nature of the MM96L cell population.After 21 days of incubation, the treated cells were seen to alignsomewhat parallel to one another in clusters, as shown in FIGS. 6C and6D, a characteristic of normal, mature melanocytes. Similar featureshave been observed with all dendritic cell-inducing fractions from E.peplus, including the crude sap.

[0120] Electrospray mass spectroscopic analyses for fractions 14 and 15indicated the presence of2,5,7,14-tetraacetoxy-3-benzoyloxy-8,15-dihydroxy-9-nicotinoyloxyjatropha-6(17),11E-diene(jatrophane 5, Jakupovic et al, 1998a) with an m/z of 780 (calculated779.315). Nuclear magnetic resonance (NMR) analysis, using 1D NMR, onfraction 14 gave down-field signals between 7 and 9.4 ppm which areconsistent with a pyridine-like moiety, as is present in the nicotinoategroup at ring position 9. Also, a trans double bond was evidenced by thelarge coupling constant at 5-6 ppm, in agreement with the 11, 12internal double bond in the jatrophane ring structure. Also identifiedin fraction 14 by electrospray in the negative ion mode was2,5,7,9,14-pentaacetoxy-3-benzoyloxy-8,15-dihydroxy-jatropha-6(17),11E-diene (jatrophane 6, Jakupovic et al, 1998a), with m/z 716(calculated 716.304), 673 (M—ketene), 656 (M—AcOH).

[0121] Fraction 15 contained2,3,5,7,15-pentaacetoxy-9-nicotinoyloxy-14-oxojatropha-6(17),11E-diene(jatrophane 1, Jakupovic et al, 1998a) with m/z 597 (M—ketene—AcOH).Thus, by spectroscopic analysis, the early-eluting fractions at 7-7.5minutes on HPLC with cell killing and dendritic activity contained amixture of jatrophanes 5, 6, and 1. This result is consistent with thebehaviour of HPLC fractions 14 and 15 when chromatographed on HPTLC,using toluene:acetone 9:1 as the developing solvent. UV-positive spotsdid not move from the origin, R_(f) 0.0 (approx), in contrast tolater-eluting fractions (eg fractions 20-22, R_(f) 0.3-0.5). Thisindicates the relatively polar behaviour of jatrophanes 5, 6, and 1, incomparison to jatrophanes 3, 2 and 4, as demonstrated by chromatographyon HPTLC, using either toluene:acetone 9:1 or hexane:ethyl acetate 4:1as developing solvents. These results are similar to those obtained byJakupovic et al, 1998a, using petrol: methyl-tert-butyl ether (1:1) asthe developing solvent, eg: jatrophane 5: R_(f) 0.04, jatrophane 6:R_(f) 0.10 (3X), and jatrophane 1: R_(f) 0.11. There was no evidence inthe mass spectroscopic data from the early HPLC fractions of thepresence of ingenane derivatives (see later), or other componentsreported from the literature and presented in Table 1, in E. pepluscrude extracts.

Example 9 Biological Activity-Guided Purification of Crude andEther-Soluble Extracts on Thin Layer Chromatography (TLC) and HighPerformance Thin Layer Chromatography (HPTLC)

[0122] (a) The ether-soluble fraction, prepared as in Example 7, wasreconstituted in ethylene glycol dimethyl ether (DME) andchromatograpned on 20×20 cm silica gel plates, using chloroform:ethylacetate (82:18) as the developing solvent (FIG. 7). The plate was viewedon a UV light box and the UV positive bands were identified, excisedfrom the gel, eluted with DME, and tested for inhibitory activity andmorphology reversal against MM96L melanoma cell line. By slicing thewhole gel into UV and non-UV absorbing fractions, it was demonstrated inpreliminary experiments that activity was associated with theUV-absorbing bands. Staining the side strips of the gel with 0.1% iodinein chloroform revealed other iodine strongly positive bands. However,these were found to possess negligible activity. UV-absorbing bands atRf 0.0 (A), Rf 0.16-0.18 (B1), Rf 0.22-0.24 (B2), Rf 0.73-0.80 (C), Rf0.80-0.96 (D) were biologically active, with observable decrease in cellnumbers and complete reversion to dendritic cell appearance at 1/5,000dilution.

[0123] Zones B1, C and D were further purified by chromatography onsilica gel 60 plates, using a two-dimensional solvent system withhexane:ethyl acetate (1:1) in the first dimension and toluene:acetone(9:1) in the second dimension (FIGS. 8A to 8C respectively).UV-absorbing spots with inhibitory activity towards MM96L of greaterthan 30% of cell numbers and with complete reversion to dendritic cellappearance at 1/500 dilution are indicated on the figures.

[0124] The strongly UV-absorbing spots 22 and 23 derived from zone D(see FIG. 8C) were excised from the gel, eluted with DME and dried byvacuum centrifugation. Mass spectroscopic analysis of fractions 22 and23 revealed the presence of5,8,9,10,14-pentaacetoxy-3-benzoyloxy-15-hydroxypepluane, m/z 639.5[M—AcOH]⁺, ie pepluane.

[0125] (b) Whole crude sap was chromatographed on 10×10 cm HPTLC silicagel 60 plates with concentrating zones (Merck Cat No. 013748.1000),using toluene:acetone (9:1) as the developing solvent, as shown in FIG.9. The UV-positive zones (1, R_(f) 0.14; 2, R_(f) 0.23; 3, R_(f) 0.49;4,R_(f) 0.54; 5, R_(f) 0.57; 6, R_(f) 0.63; and 7, R_(f) 0.73) wereexcised from the gel and eluted with DME/diethyl ether. The fractionswere tested against MM96L as described above, and fractions 1, 3, 4, 5and 6 were demonstrated to possess cell inhibitory activity and cellreversion activity. These fractions were separately chromatographed onHPTLC plates using hexane : ethyl acetate (4:1) as the developingsolvent, yielding UV positive bands A, R_(f) 0.17; B, R_(f) 0.24; C,R_(f) 0.42; D, R_(f) 0.48; E, R_(f) 0.52; F, R_(f) 0.58; G, R_(f) 0.62(FIG. 10) and H, R 0.02 (FIG. 11). All fractions except G (iodinepositive, see FIG. 10) were active against MM96L cells, in terms of cellgrowth inhibition and reversion to complete dendritic morphology, at 1in 5000 dilution.

[0126] Mass spectroscopic analyses of fractions A-F (B missing) and Hare shown in Table 15, with a tentative assignment of compounds from theknown molecular mass ions of the published constituents of E. peplus:TABLE 15 Mass spectroscopic analysis of HPTLC Fractions Fraction m/z,Relative Abundance (%) and tentative assignment A 495.2357 (100)[C₂₇II₃₆O₇Na⁺ (ingenol acetate)], 433.3799 (51), 579.2916 (39)[pepluane - 2AcOH]⁺, 679.2754 (16), 691.4046 (16) B N.D. C 579.2846(100) [pepluane - 2AcOH]⁺, 691.4073 (50), 747.47 (8) [jatrophane 3 -AcOH]⁺, 803.53 (11) D 579.2827 (100) [pepluane - 2AcOH]⁺, 691.4025 (23),715.3686 (38) [jatrophane 2 - ketene]⁺ E 437.2254 (100), 619.5287 (18),[jatrophane 4, 638 - ketene + Na⁺], 647.5615 (18) [jatrophane 4 -2AcOH + Na⁺] F 591.4996 (55), 619.5299 (100) [jatrophane 4, 638 -ketene + Na⁺], 647.5635 (77) [jatrophane 4 - 2AcOH + Na⁺], 691.4183 (49)H 830.3216 (100) (jatrophane 3 + Na⁺]

[0127] pepluane=5,8,9,10,14-pentaacetoxy-3-benzoyloxy-15-hydroxypepluane

[0128] jatrophane2=2,5,7,9,14-hexaacetoxy-3-benzoyloxy-15-hydroxy-jatropha-6(17),11E-diene

[0129]jatrophane3=2,5,14-triacetoxy-3-benzoyloxy-8,15-dihydroxy-7-isobutyroyloxy-9-nicotinoyloxyjatropha-6(17),11E-diene

[0130] jatrophane4=2,5,9,14-tetraacetoxy-3-benzoyloxy-8,15-dihydroxy-7-isobutyroyloxyjatropha-6(17),11E-diene)

[0131] Thus, mass spectroscopy revealed a mixture of20-acetyl-ingenol-3-angelate (fraction A), pepluane (fractions A, C & D)and jatrophanes 2 (fraction D) 3 (fractions C&H), and 4 (fractions E &F). ¹H chemical shift data for fraction H are shown in Table 16. TABLE16 ¹H Chemical Shift* Data for Fraction H H ppm Multiplicity IndicativeJatrophane Ring Backbone Signals  1α 2.816 brd  1β 2.056 d  3 5.918 d  43.731 brd  5 5.730 brd  7 5.390 d  8(OH) 2.948 d  9 4.971 s 11 6.145 d12 5.640 dd 13 2.840 cm 14 5.110 s 15(OH) 3.645 s 16 1.489 s 17 4.438 d17′ 4.788 d 18 1.052 s 19 1.152 s 20 1.353 d Ester Substituent SignalsOnic 9.290 brd 8.340 ddd 8.805 brdd 7.390 brd 9.079 brd Onic 8.202 ddd8.767 brdd 7.327 brd 8.040 AA′ OBz 7.403 BB′ 7.541 C 1.972 qq OiBu 0.912d 0.449 d 0.450 d

[0132] These assignments indicated the presence of a jatrophane ringstructure as determined from DQF-COSY, NOESY and TOCSY two-dimensionalspectra. The spectrum of Fraction H was consistent with the presence ofJatrophane 3 in two diastereomeric conformations (considered mostlikely), a mixture of two or more similarly substituted jatrophanes, ora new jatrophane with two nicotinate, one benzoate, and an iso-butyratemoiety. The likely ring confirmation was II, as per Jakupovic et al(1998a), with a J4,5 of approximately 6 Hz and strong NOE's between 5and 8, and 4 and 7; with J7,8 and J8,9 practically zero—as evidenced bytotal lack of cross peaks in the DQF COSY spectrum. There were nosignals consistent with the presence of any ingenol structure. Thesample was retrieved from the magnet, and an aliquot demonstrated potentactivity against MM96L, evidenced by complete cell death at 20 μg/ml,and complete reversion to a dendritic appearance at less than 20 pg/ml.

Example 10 NMR Analysis

[0133] Fraction A was further purified by chromatography on HPTLC usinghexane:ethyl acetate (4:1) as the developing solvent. As an adjunct toabsorbance on a UV light box, a side strip was stained by spraying thegel with 70% phosphoric acid in methanol, and development by heating thegel with a hair drier revealed an intense blue band under UV light,separable from the major UV absorbing band. The unstained regionequivalent to this band was excised, eluted with ether and dried byvacuum centrifugation. Approx. 1 mg of this material was accumulatedfrom 4 ml latex. The material was subjected to NMR analysis, andsubsequently bioassayed and demonstrated to be active in terms ofreversion to complete dendritic morphology at 1 in 5×10⁶ dilution,representing a 1 ng/ml final concentration. This material was identifiedby NMR as C₂₇H₃₆O₇, 20-acetyl-ingenol-3-angelate as shown in Table 17.This finding is consistent with the mass spectroscopic evidencepresented in Table 15. TABLE 17 NMR data obtained on bioactive fractionA2 to support 20-acetyl-ingenol-3-angelate chemical structure: ¹H NMR¹³C NMR H ppm/multiplicity # C Hz [PPM]  1 6.106  1 19 25933.893206.2210  3 5.396 s  2 26 21513.354 171.0737  5 3.875 d  3 21 21165.912168.3070  7 6.024 d  4 23 17626.074 140.1539  8 4.076  5  2 17086.833135.8710 11 2.4733 m  6 16 17032.062 135.3330 12 2.222 ddd  7  116614.730 132.1169 12′ 1.743 ddd  8  7 16301.014 129.6223 13 0.681 m  922 15976.620 127.0428 14 0.936 m 10 14 10668.691 84.8352 16 1.033 s 11 3 10395.504 32.6629 17 1.062 S 12  5 9411.636 74.8398 18 0.952 d 13 109059.148 72.0365 19 1.785 brs 14 20 8404.062 66.8274 20 4.745 d 15  85481.636 43.5892 20′ 4.467 d 16 11 4841.115 38.4955 23 6.153 gg 17 123911.906 31.1067 24 1.906 brs 18 ? 3735.577 29.7045 25 1.996 brdd 19 163585.756 28.5132 27 2.042 20 15 3018.427 24.0019 40H 3.4308 21 132924.303 23.2535 50H 3.514 d 22 14 2392.363 23.0035 23 27 2655.73431.1179 24 24 2612.189 20.7716 25 18 2171.913 17.2706 26 25 2007.76015.9653 27 19 1698.690 15.6546 28 17 1931.372 15.5169

[0134] However, the absence of 20-acetyl-ingenol-3-angelate from themass spectra of the activity-guided purifications by HPLC, and in otherTLC fractions apart from fraction A, indicates that this is not the onlyactive fraction. Rather, jatrophanes 1-6 and pepluane are alsoimplicated by deduction from the NMR and mass spectroscopic data. Thisis particularly true of fractions H as prepared by TLC (jatrophane 3 Na⁺m/z 830; see also 1D NMR results in Table 16) and fractions 13 and 14 asprepared by HPLC (jatrophane 5, m/z 779 and 1D NMR; jatrophane 6, m/z716; jatrophane 1 or jatrophane 6 derivative, m/z 597.

[0135] Jakupovic et al (1998a) have proposed that the paraliane class ofcompounds are intermediates in the pathway between jatrophanes andpepluane. Since anti-cancer cell activity and dendritic cell reversal byboth jatrophanes and pepluane have been demonstrated in this invention,it seems reasonable to conclude that the paralianes will also exhibitthese properties.

Example 11 Preparation of Material for the Mouse Experiments byPreparative Thin Layer Chromatography

[0136] 15 ml crude sap in 70% ethanol was extracted with diethyl etheras described in Example 6. The extract was concentrated by vacuumcentrifugation and resuspended in approx 5 ml DME. The DME extract waschromatographed on preparative TLC plates (Merck PLC, Silica gel 60, Catno. 005745.1000) using hexane:ethyl acetate (4:1) as the developingsolvent. Zones corresponding to regions “H” and “A-F” as shows in FIG.12 were excised and combined, eluted with ether/DME, and dried by vacuumcentrifugation. The extract was enriched in jatrophanes 2, 3 and 4,pepluane, and the ingenane acetate. The pellet was suspended in 95%ethanol and centrifuged at 10,000 g for 10 minutes. The supernatant (6.0ml, 10 mg/ml) was distributed into 0.2 ml aliquots and stored at −20° C.This extract was assayed against MM96L melanoma cell line, and showedhigh potency, with dendritic cell morphology still evident at 1 in 5×10⁶dilution; this replicated the potency of the crude sap. The extract soprepared was enriched in jatrophanes 2, 3 and 4, pepluane, and theingenane acetate. Just prior to injection, 20 μl was diluted to 1 mlwith RPMI-1640 tissue culture medium containing 5% foetal calf serum forinjection of 0.1-0.2 ml. The ethanol solution (10 mg/ml) was absorbed ona cotton bud (0.2-0.4 ml) and used for topical application in mice.

Example 12 Inhibition of Growth of Subcutaneous Implants of Tumour Cells

[0137] (a) Five 4 week old nude mice were injected s.c. at 4 differentsites with 0.1 ml of tissue culture medium containing 2×10⁶ MM96L humanmelanoma cells. The three treated mice were injected on days 1, 2, 3, 5,6, 7, and 8 with 0.1 ml RPMI medium containing 5% foetal calf serum and20 μg ethanol extract. In addition, the treated mice received up to fourtopical applications of approx 5-10 μl of 10 mg/ml ethanol extract orcrude undiluted sap. Two separate sites on each treated mouse receivedtopical treatment with either ethanol extract or crude sap. One mousereceived topical treatment on days 12, 13 and 14, and the other twotreated mice received topical treatment on days 15, 19, 20 and 22.Tumour volume was measured on day 32.

[0138] Prior to the topical applications, injection of extract had noapparent effect on tumour volume. Following topical application ofethanol extract there was an overnight change in tumour appearance. Thetumours became greyish-black in colour, then developed a hard, lumpyblack appearance followed by scab formation. Tumours treated with crudesap showed similar changes a day later. With time, the overall effectsof ethanol extract and crude sap were similar, so measurements for thetopically treated lesions have been combined. On the mice giveninjection plus topical treatment, tumour volume was reduced by 76%(p<0.2). One tumour which had been treated with the ethanol extract hadcompletely disappeared, as shown in FIG. 13, and eight others werereduced to flat black scabs. The other three treated tumours initiallyshowed similar colour changes and tumour regression, but had regrownfollowing cessation of topical treatment 10 days before the measurementswere taken.

[0139] (b) Six 4 week old C57 Black (C57B1) mice were injected with 0.1ml of tissue culture medium containing 10⁵ B16 melanoma cancer cells attwo sites on the underbelly. The tumours were allowed to develop for 4days, and then were subjected to a regimen of three injections (20 μgethanol extract in 0.1 ml RPMI medium containing 5% foetal calf serum(days 1, 2 and 4) and 1 topical treatment (5-10 μl of 10 mg/ml ethanolextract on day 4). 8 days after the first injection the areas of thetumours were measured using a ruler. Treatment reduced the size of theB16 melanoma tumours by 64% (p<0.05) on the three treated mice bycomparison with the size of tumours on the three control mice.

[0140] The results are summarised in Table 18. TABLE 18 Inhibition ofTumour Growth In Vivo by E. peplus Extracts Treatment No of Tumour size*Model regimen tumours control treated % inhibition MM96L human melanoma(a) 12 89.8 ± 37  21.5 ± 3.6  76 cell line, on nude (p < 0.20) mice B16mouse melanoma (b)  6 58.5 ± 9.5 21.2 ± 10.6 64 on C5781 Black mice (p <0.05)

Example 13 Changes in Gene Expression Induced in a Human Melanoma CellLine (MM96L) by Purified Extract

[0141] Human melanoma cells of the MM96L cell line, cultured in 150 cm²plates in RPMI 1640 medium containing 10% foetal calf serum, wereincubated with purified extract for 4 hr at 37° C. in 5% CO₂/air. Cellswere washed with phosphate buffered saline (PBS), scraped in PBS,pelleted, resuspended in 1 ml PBS, pelleted and taken up in 300 μl NP-40lysis buffer, left on ice for 15 min, pelleted and the supernatanttreated with proteinase K and SDS at 37° C. for 15 min, extracted withphenol chloroform and the total RNA precipitated by ammoniumacetate/ethanol at −20° C. overnight. The Promega mRNA isolation kit wasused to isolate mRNA, which was then reverse transcribed in the presenceof ³³P-labelled dCTP to generate cDNA. The latter was hybridised on aGenome Systems human Gene Discovery Array 1.2 (GDA) according to themanufacturer's instructions. The array was quantitated with a MolecularDynamics PhosphorImager, and analysed with ImageQant and Excel software.

[0142] The ratio of duplicate spot volumes from treated and untreatedcells was calculated, and used to define the level of gene activation(ratio >1) or inhibition (<1). Backgrounds were typically 500-1000counts, but were not subtracted; thus the stated ratios will tend to beunderestimates of the actual changes.

[0143] The array contained cDNA spots from over 18,000 unique sequences,so-called expressed sequence tags (ESTs), of which approximately 3000were from identifiable expressed genes of human cells. Many ESTsequences in the human melanoma cells tested were either up- ordown-regulated by the extract treatment. Only changes based onduplicates which had standard deviations <30% of the ratio wereconsidered to be biologically significant at this stage. It should alsobe noted that a relatively short treatment time of 4 hr was used inorder to identify the earliest and most critical targets for the agent.It is likely that further, major changes in gene expression, dependentupon the primary response, will occur after this time.

[0144] Results from the changes in level of the transcripts of somerelevant known genes, considered to be beneficial either directly orindirectly for the control of cancer cells, are summarized in Table 19.

[0145] The changes in cell morphology observed in the Examples can beexpected to result from the major down-regulation of a number ofproteins that bind to actin, a major cytoskeletal protein. An increasein the retinol binding protein may also be involved here, as well as ininduction of the differentiated phenotype through increasing theintracellular level of retinoids.

[0146] Repair of current and future DNA damage induced by solar UVirradiation may be enhanced by the observed induction of XP repairproteins. In addition, the decrease in GADD45 and ionising radiationresistance protein (DAP3) may be useful in sensitising tumour tissue toradiotherapy. The latter change is also notable because it is stronglyupregulated in MM96L cells by UVB, the cause of skin cancer andmelanoma.

[0147] A number of molecules relevant to enhancing the immune responsewere induced, particularly G-CSF. Some of these, such as proteins of themajor histocompatability complex (MHC), are considered to be usefulattributes for immunotherapy, enhancing killer T-cell activity.

[0148] The changes most significant for control of cell growth relate tothe detected alterations of the G-protein and PKC pathways, andenhancement of proteosome activity. Intracellular signalling is criticalfor many cell processes, including proliferation and alterations in thenormal equilibrium of pathways and pathway interactions, such as thosemediated by Ras signalling,are likely to have adverse consequences forthe cell. The level of induction of the proteosome component LAMP7-E1was among the highest found for any gene in the experiment, and would beexpected to greatly alter the processing of many proteins via theubiquitin pathway.

[0149] On the basis of the gene expression array data, the compounds ofthis invention are expected to have activity:

[0150] 1. In modulating gene expression in the G-protein, PKC and Rassignalling pathways, in a manner that leads to anticancer activity invivo.

[0151] 2. In ameliorating damage from solar UV and like agents, byenhancing DNA repair and the immune response, either in the target oreffector cells.

[0152] 3. As an adjuvant to radiotherapy or to therapy with otherDNA-damaging agents, on the basis of down-regulation of protectiveproteins (GADD45 and DAP3). TABLE 19 Regulation Function Gene EGAD no.by Extract Reference Immune response Sialyltransferase MHC class 1HT4978 2.16 Li et al, 1998 proteins HT3059 2.64 G-CSF receptor HT26801.39 HT4313 11.68 Cell growth 80H-K HT1772 2.11 Kanai et al, regulationFibroblast growth factor 9 HT2447 0.59 1997 Differentiation Cellularretinol binding HT2520 2.69 Perozzi et al, protein 1 1998 C-protein Betapolypeptide 3 HT484  2.27 pathways G-binding protein HT3752 0.35 Small Gprotein TTF HT5016 0.47 PKC pathways Phospholipase D HT2473 4.04 Boschet al, PKC zeta HT21136 0.67 1998 Tumour Wilm's tumour-related proteinHT3751 1.99 suppressor genes DNA damage and XP group C p58 HT4209 2.36repair proteins Hsp 27/28 HT2997 2.36 XP group C HHR2 HT4247 2.09 GADD45HT3135 0.63 Ionising radiation resistance HT5168 0.46 protein (DAP3)Proteolysis LAMP7-E1 HT3850 26.91 Mimnaugh et al, 1997 Cell morphologyProfilin II HT928  0.62 Djafarzadeh, Cofilin HT1657 0.56 1997Cyclophilin B HT1953 0.36 Tubulin alpha k1 HT1813 0.61 Oncogenes TAXHT3360 0.32 Pise-Masison et al, 1998

Example 14 Treatment of a Solar Keratosis in a Human Volunteer

[0153] Ethics committee approval was obtained from the QueenslandInstitute of Medical Research for a clinician-supervised trial of use ofcrude sap of E. peplus for treatment of a facial solar keratosis in ahuman subject.

[0154] Crude extract obtained from Australian-grown plants and stored in50% glycerol for 2 weeks at −20° C. was applied with a cotton budapplicator to the surface of a clinically diagnosed solar keratosis,approximately 5 mm in diameter, on the left temple of the face of a malehuman volunteer. Approximately 50 μl was delivered to the surface. Oneday later, a second application was made to the same site. After thefirst application, no reaction was noted for 4-5 h, whereafter aninflammation reaction occurred at the site and extended to an area of80-100 mm in diameter. One day later, there was localised swelling, andblister formation at the site of application and on localised patchesdistal to the area of application, as if new premalignant sites werealso targeted. After four days following the first treatment, theswelling subsided and scab formation was evident at the affected sites.After fourteen days, the scabs had sloughed off, leaving new skinunderneath. After six weeks, the treated areas still had a pinkishtinge, but there was no sign of the original solar keratosis. As acontrol, a 1 cm² patch of normal skin on the forearm of the samevolunteer was similarly treated. There was localised mild inflammation,which disappeared 7-10 days after treatment.

[0155] The strong inflammatory reaction associated with treatment of thesolar keratosis could reflect recruitment and proliferation of killer-Tcells, as suggested by the results for immune response obtained from thegene array screen in Example 13, and the observation of in vitroproliferation of T-cells by E. peplus crude sap in Example 15 below.Enhancement of killer-T cell activity is considered to be a key step indestruction of cancer cells by the immune system and may help to explainthe recognition and attack of premalignant lesions distal to the site oforiginal treatment.

Example 15 Effect of Crude Sac and Purified Fractions “A” and “H” fromTLC on Normal Melanocyte Cell Numbers

[0156] 12-O-tetradecanoylphorbol-13-acetate (TPA) is essential for theculture of normal melanocytes in vitro, since these cells grow verypoorly without TPA. In a preliminary experiment, E. peplus fractionswere added to medium without added TPA from the start of the experiment.E. peplus fractions were added to fresh medium, and the cell numbersscored compared to fresh media without E. peplus fractions or TPA. Underthis regimen, higher numbers of melanocytes were obtained than with the“control” cells grown in TPA-deficient medium. Interestingly, the cellsin the medium with E. peplus fractions looked healthier than those cellsgrown in so-called “standard” medium with TPA. Thus E. peplus-derivedcompounds may provide a superior alternative to the use of TPA as a toolin cell culture.

[0157] In a second experiment, normal melanocytes were plated at 5000cells per well, in RPMI 1640 medium containing 10% foetal calf serum,cholera toxin, antibiotics, and TPA. After 24 hours, the medium wasremoved from the cells by suction, and replaced with fresh mediumwithout added TPA, but with the additions as specified. Cells werescored after a further 10 days of incubation. The results are shown inTable 20. It is evident that even at a 1 in 5,000,000 dilution a cellproliferation effect was noted with crude and purified fractions, incontrast to cell inhibitory effects observed at these concentrationsagainst cancer cell lines as shown in earlier examples. In a separatetest, in vitro proliferation of T cells was also obtained followingtreatment of T cells with crude E. peplus sap. TABLE 20 Sample 1/501/500 1/5,000 1/50,000 1/500,000 1/5,000,000 Solvent(control) + + + + + + crude E. peplus sap − + + ++ ++ ++ Fraction “A”, +++ ++ ++ ++ + (enriched in ingenol acetate) Fraction “H”, ± ++ ++ ++ ++++ (enriched in jatrophane 3)

[0158] Since both normal melanocytes and T-cells were induced toproliferate by fractions from E. peplus sap, this agent may have wideapplication as a cell proliferation agent for normal cells, either invivo or in vitro, in any medical condition where regeneration of normalcells would be advantageous, including but not limited to

[0159] a) multiplication of skin cells (keratinocytes) for rapid woundhealing in trauma cases and after surgery, and in recovery from burns.

[0160] b) multiplication of pancreatic islet cells for implantation

[0161] c) multiplication of T-cells and other cells of the immunesystem. It is interesting to note that the expansion of action past thepoint of application in the human volunteer trial on treatment of solarkeratosis may be explained by a recruitment of natural killer-T cells tothe region of application.

[0162] d) regeneration of aged or necrotic tissue from liver, kidney,colon, lung and eye.

[0163] e) multiplication of host tissue as an alternative to organtransplantation

Example 16 Effect of Betaines on Malignant Melanoma MM96L Cell Numbers

[0164] Betaines of different types were solubilised in sterile MilliQ™water to a final concentration of 1 mg/ml, and diluted into 0.1 mltissue culture medium containing 5000 MM96L cells as describedpreviously. Cells were scored after 4 days incubation. The results areshown in Table 21.

[0165] Whereas most betaines tested had no effect on cell numbers,β-alanine betaine hydrochloride (homobetaine) depressed cell numbers ata final concentration of 20 μg/ml, and the cells had a dendriticappearance. t-4 hydroxy N,N-dimethyl proline also inhibited cell numbersat a final concentration of 20 μg/ml; however, the cell morphologychanged to that of polydendritic forms, the significance of which isunknown.

[0166] It is envisaged that β-alanine betaine hydrochloride(homobetaine) may be a suitable formulation agent for E. peplus crudesap or its purified active principles, including ingenol, pepluane, andjatrophanes 1-6, either separately or in combination. This could be usedfor topical application against premalignant skin lesions at lowdilutions of E. peplus principle(s), or formulated as an anticancer drugwith higher concentrations of E. peplus principle(s). it has beensuggested that betaines per se are useful as anti-cancer agents; see forexample U.S. Pat. No. 5,545,667 by Wiersema et al.

[0167] Because of their surfactant properties, betaines are widely usedas formulation ingredients in cosmetics. Due to their zwitterionproperties, betaines could also assist transport of other ingredientsinto the deeper layers of the skin. A betaine to be used in a skincosmetic preparation along with very dilute extracts of E. peplus sap orpurified fractions derived therefrom, such as jatrophanes, pepluane,paraliane, or ingenane, separately or in combination, should desirablyhave complementary properties. Of all the betaines tested, includingglycine betaine, only β-alanine betaine hydrochloride (homobetaine) hada phenotype reversal effect, albeit modest, as compared to E. peplus sapand fractions. TABLE 21 Sample 1/50 1/500 1/5,000 glycine betaine ++++++ +++ N-methyl proline, free base +++ +++ +++ t-4-hydroxy N-methylproline, free +++ +++ +++ base stachydrine (proline betaine), +++ ++++++ free base t-3-hydroxy N-methyl proline, free +++ +++ +++ baseβ-alanine betaine hydrochloride ++d +++ +++ (homobetaine) t-4 hydroxyN,N-dimethyl proline, +pd +++ +++ free base

[0168] It will be apparent to the person skilled in the art that whilethe invention has been described in some detail for the purposes ofclarity and understanding, various modifications and alterations to theembodiments and methods described herein may be made without departingfrom the scope Of the inventive concept disclosed in this specification.

[0169] References cited herein are listed on the following pages, andare incorporated herein by this reference.

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1. A compound present in plants of the genus Euphorbia, and inparticular in sap of Euphorbia peplus, Euphorbia hirta and/or Euphorbiadrummondii, which: (a) is able to kill or inhibit the growth of cancercells, but does not significantly affect normal neonatal fibroblasts, orspontaneously transformed keratinocytes; (b) has activity which is notdestroyed by heating at 95° C. for 15 minutes; (c) has activity which isnot destroyed by treatment with acetone; (d) has activity which can beextracted with 95% ethanol; and (e) stimulates metallothionein geneactivation.
 2. A compound according to claim 1 , which is able toinhibit the growth of at least one cell line selected from the groupconsisting of MM96L, MM229, MM220, MM537, MM2058, HeLa, B16, LIM1215,A549, MCF7, MCC16 and Colo16, as herein defined.
 3. A compound accordingto claim 2 , which is able to inhibit growth of or to inducedifferentiation in MM96L cells.
 4. A compound according to any one ofclaims 1 to 3 , which is also able to induce normal melanocytes and/or Tcells to proliferate.
 5. A composition comprising a compound accordingto any one of claims 1 to 4 , together with a pharmaceutically- orcosmetically-acceptable carrier.
 6. A composition according to claim 5 ,in which the compound is selected from the group consisting ofjatrophanes, pepluanes, paralianes and angeloyl-substituted ingenanes oracetylated derivatives thereof, and pharmaceutically-acceptable salts oresters thereof.
 7. A composition according to claim 6 , in which thecompound is a jatrophane of Conformation II.
 8. A composition accordingto claim 6 or claim 7 , in which the compound is substituted as definedin Table
 2. 9. A composition according to any one of claims 5 to 8 , inwhich the compound is selected from the group consisting of:5,8,9,10,14-pentaacetoxy-3-benzoyloxy-15-hydroxypepluane (pepluane);15-pentaacetoxy-9-nicotinoyloxy-14-oxojatropha-6(1),11E-diene(jatrophane 1);2,5,7,9,14-hexaacetoxy-3-benzoyloxy-15-hydroxy-jatropha-6(17),11E-diene(jatrophane 2);2,5,14-triacetoxy-3-benzoyloxy-8,15-dihydroxy-7-isobutyroyloxy-9-nicotinoyloxyjatropha-6(17),11E-diene(jatrophane 3);2,5,9,14-tetraacetoxy-3-benzoyloxy-8,15-dihydroxy-7-isobutyroyloxyjatropha-6(17),11E-diene)(jatrophane 4);2,5,7,14-tetraacetoxy-3-benzoyloxy-8,15-dihydroxy-9-nicotinoyloxyjatropha-6(17),11E-diene(jatrophane 5);2,5,7,9,14-pentaacetoxy-3-benzoyloxy-8,15-dihydroxyjatropha-6(17),11E-diene(jatrophane 6); 20-acetyl-ingenol-3-angelate; andpharmaceutically-acceptable salts or esters thereof.
 10. A compositionaccording to any one of claims 5 to 9 , in which the carrier comprisesβ-alanine betaine hydrochloride or t-4-hydroxy-N,N-dimethyl proline. 11.A method of treatment of a cancer, comprising the step of administeringan anti-cancer effective amount of a compound according to any one ofclaims 1 to 4 or a composition according to any one of claims 5 to 10 toa mammal in need of such treatment.
 12. A method according to claim 11 ,in which the cancer is a solid tumour.
 13. A method according to claim11 or claim 12 , in which the cancer is selected from the groupconsisting of malignant melanoma, other skin cancers including Merkelcell carcinoma, squamous cell carcinoma and basal cell carcinoma, lungcancer, colon cancer, prostate cancer, cervical cancer and breastcancer.
 14. A method according to any one of claims 11 to 13 , used asan adjuvant to radiotherapy or to therapy with a DNA-damaging agent. 15.A method of inhibiting proliferative activity of neoplastic cells,comprising the step of exposing the cells to an anti-proliferativeamount of a compound according to any one of claims 1 to 4 , or acomposition according to any one of claims 5 to 10 .
 16. A method ofstimulating proliferation of non-neoplastic cells, comprising the stepof exposing the cells to a proliferation-inducing amount of a compoundaccording to any one of claims 1 to 4 or a composition according to anyone of claims 5 to 10 .
 17. A method of stimulating the activity and/orinducing proliferation of T-cells, comprising the step of exposing thecells to an effective amount of a compound according to any one ofclaims 1 to 4 , or a composition according to any one of claims 5 to
 10. 18. A method of alleviating disease conditions by stimulating cells ofthe immune system, according to claim 16 .
 19. A method of inducingneoplastic cells to differentiate, comprising the step of exposing thecells to an effective amount of a compound according to any one ofclaims 1 to 4 , or a composition according to any one of claims 5 to
 10. 20. A method according to any one of claims 15 to 18 , in which thecells are treated in vivo.
 21. A method according to any one of claims15 to 18 , in which the cells are treated ex vivo.
 22. A method ofpreventing or alleviating damage to skin caused by ultravioletirradiation, ionizing radiation, microwave radiation or exposure toozone, comprising the step of topically administering an effectiveamount of a compound according to any one of claims 1 to 4 or acomposition according to any one of claims 5 to 10 to a subject in needof such treatment.
 23. A method according to claim 22 , in which thedamage is solar keratosis.
 24. A method according to claim 22 , in whichthe damage occurs during radiotherapy.
 25. A method according to any oneof claims 11 to 24 , in which the mammal is a human.
 26. Use of acompound according to any one of claims 1 to 4 in the treatment ofcancer.
 27. Use of a compound according to any one of claims 1 to 4 forpreventing or alleviating damage to skin caused by ultravioletirradiation, ionizing radiation, microwave radiation or exposure toozone.
 28. Use of a composition according to any one of claims 5 to 10in the treatment of cancer.
 29. Use of a composition according to anyone of claims 5 to 10 in preventing or alleviating damage to skin causedby ultraviolet irradiation, ionizing radiation, microwave radiation orexposure to ozone.
 30. Use of a compound according to any one of claims1 to 4 , or a composition according to any one of claims 5 to 10 in themanufacture of a medicament for the treatment of cancer.
 31. Use of acompound according to any one of claims 1 to 4 , or a compositionaccording to any one of claims 5 to 10 in the manufacture of amedicament for preventing or alleviating damage to skin caused byultraviolet irradiation, ionizing radiation, microwave radiation orexposure to ozone.
 32. Use of compound according to any one of claims 1to 4 , or a composition according to any one of claims 5 to 10 in themanufacture of a medicament for inducing proliferation of non-neoplasticcells.